Search

Inclusive of 20 chapters subdivided into 4 parts, this book's second edition covers latest updates in the current understanding of the molecular biology, biochemistry and immunology of parasitic nematodes of humans, domestic animals and plants. Specific topics discussed in each chapter include horizontal gene transfer, immune expulsion mechanisms, genetics of human susceptibility to nematode infections, nematode protein structure, nematode bacterial symbionts, and host immune system modulation by nematode parasites and their therapeutic potential for immunological diseases.

Among many survival strategies, parasitic worms secrete molecules that modulate host immune responses. One such product, ES-62, is protective against collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Since interleukin-17 (IL-17) has been reported to play a pathogenic role in the development of RA, this study was undertaken to investigate whether targeting of IL-17 may explain the protection against CIA afforded by ES-62.DBA/1 mice progressively display arthritis following immunization with type II collagen. The protective effects of ES-62 were assessed by determination of cytokine levels, flow cytometric analysis of relevant cell populations, and in situ analysis of joint inflammation in mice with CIA.ES-62 was found to down-regulate IL-17 responses in mice with CIA. First, it acted to inhibit priming and polarization of IL-17 responses by targeting a complex IL-17-producing network, involving signaling between dendritic cells and γ/δ or CD4+ T cells. In addition, ES-62 directly targeted Th17 cells by down-regulating myeloid differentiation factor 88 expression to suppress responses mediated by IL-1 and Toll-like receptor ligands. Moreover, ES-62 modulated the migration of γ/δ T cells and this was reflected by direct suppression of CD44 up-regulation and, as evidenced by in situ analysis, dramatically reduced levels of IL-17-producing cells, including lymphocytes, infiltrating the joint. Finally, there was strong suppression of IL-17 production by cells resident in the joint, such as osteoclasts within the bone areas.Our findings indicate that ES-62 treatment of mice with CIA leads to unique multisite manipulation of the initiation and effector phases of the IL-17 inflammatory network. ES-62 could be exploited in the development of novel therapeutics for RA.

Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition.

Helminths may protect humans against allergic and autoimmune diseases and, indeed, defined helminth-derived products have recently been shown to prevent the development of such inflammatory diseases in mouse models. Here, we propose that helminth-derived products not only have therapeutic potential but can also be used as unique tools for defining key molecular events in the induction of an anti-inflammatory response and, therefore, for defining new therapeutic targets.

SummaryThe dramatic recent rise in the incidence of allergic or autoimmune inflammatory diseases in the West has been proposed to reflect the lack of appropriate priming of the immune response by infectious agents such as parasitic worms during childhood. Consistent with this, there is increasing evidence supporting an inverse relationship between worm infection and T helper type 1/17 (Th1/17)-based inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes and multiple sclerosis. Perhaps more surprisingly, given that such worms often induce strong Th2-type immune responses, there also appears to be an inverse correlation between parasite load and atopy. These findings therefore suggest that the co-evolution of helminths with hosts, which has resulted in the ability of worms to modulate inflammatory responses to promote parasite survival, has also produced the benefit of protecting the host from pathological lesions arising from aggressive proinflammatory responses to infection or, indeed, aberrant inflammatory responses underlying autoimmune and allergic disorders. By focusing upon the properties of the filarial nematode-derived immunomodulatory molecule, ES-62, in this review we shall discuss the potential of exploiting the immunomodulatory products of parasitic worms to identify and develop novel therapeutics for inflammation.

ES-62 is a protein that is actively secreted by filarial nematodes during parasitism of the vertebrate host. The molecule is able to directly interact with a number of cells of the immune system including B-lymphocytes, dendritic cells, macrophages and mast cells. Interaction appears to be dependent on complexing with TLR4 and results in modulation of the activity of a number of signal transduction molecules including MAP kinases, PI-3 kinase and NF-kappaB. Immunomodulatory activity of ES-62 appears to be largely due to the presence of phosphorylcholine (PC) moieties covalently attached to N-type glycans. The net effect of ES-62's interaction with the immune system is the generation of an anti-inflammatory immunological phenotype. As a consequence of this, ES-62 demonstrates striking drug-like activity in models of disease associated with aberrant inflammation, in particular those associated with autoimmunity and allergy.

The filarial nematode product ES-62 contains phosphorylcholine (PC) covalently attached to N-linked glycans. ES-62 induced high levels of immunoglobulin G1 (IgG1) antibodies, but no IgG2a, to non-PC epitopes of the molecule following subcutaneous injection into BALB/c mice. Conversely, mice given ES-62 lacking PC demonstrated significant production of both IgG subclasses. Thus, PC appears to block production of IgG2a antibodies to other epitopes on the parasite molecule. A role for interleukin-10 (IL-10) in this effect was shown by the ability of IL-10−/−mice to make an IgG2a antibody response to non-PC epitopes of ES-62.

Helminth infections are generally characterized by dominant T-helper type 2 (Th2) immune response polarization and have been shown in some cases to modulate immune responses to vaccines, i.e., the Bacillus Calmette-Guerin (BCG) vaccine. The filarial nematode secreted product ES-62 has been shown to possess immunomodulatory activities, such as the ability to inhibit pro-inflammatory/Th1 immune responses and to have therapeutic potential against diseases associated with such responses. This study aimed to investigate the ability of ES-62 to modulate the immune response to purified protein derivative (PPD), a component of the BCG vaccine designed to provoke a Th1 response. Overall, the results show that ES-62 was not capable of modulating the Th1 immune response induced by PPD, demonstrating that the helminth product, if employed therapeutically, is unlikely to interfere with the protective effects of the vaccine.

ES-62 is a protein that is actively secreted by filarial nematodes during parasitism of the vertebrate host. The molecule is able to direcdy interact with a number of cells of the immune system including B-lymphocytes, dendritic cells, macrophages and mast cells. Interaction appears to be dependent on complexing with TLR4 and results in modulation of the activity of a number of signal transduction molecules including MAP kinases, PI-3 kinase and NF-κB. Immunomodulatory activity of ES-62 appears to be largely due to the presence of phosphorylcholine (PC) moieties covalendy attached to N-type glycans. The net effect of ES-62’s interaction with the immune system is the generation of an antiinflammatory immunological phenotype. As a consequence of this, ES-62 demonstrates striking drug-like activity in models of disease associated with aberrant inflammation, in particular those associated with autoimmunity and allergy.

A 14-kDa antigen present on the surface of uterine microfilariae of Onchocerca spp. has been identified using monoclonal antibodies. The antigen was also found in skin microfilariae, but in a masked or cryptic form. A complementary DNA clone encoding the epitope recognised by one of the monoclonal antibodies was identified in a lambda gt11 library. Nucleotide sequencing revealed that the 233-bp cDNA fragment codes for the carboxy-terminus of the antigen. The deduced amino acid sequence consists of three hydrophobic domains with high potential for beta-sheet formation. The amino-terminal hydrophobic domain is followed by 4 positively charged residues (positions 22-25) which contribute to the rather basic character of the protein. Another interesting feature of the polypeptide is its richness in phenylalanine (12.7%). From the sequence information, a synthetic peptide was synthesised which was recognised by one of the monoclonal antibodies directed against the 14-kDa antigen and a small number of sera from patients with onchocerciasis. The relevance of this to vaccination is discussed.

There has been an alarming increase in the incidence of autoimmune and allergic diseases in Western countries in the past few decades. However, in countries endemic for parasitic helminth infections, such diseases remain relatively rare. Hence, it has been hypothesised that helminths may protect against the development of autoimmunity and allergy. This article reviews the evidence supporting this idea with respect to helminths of the phylum Nematoda (nematodes), considering data from human studies and animal models of inflammatory disease. The nature and mode of action of nematode-derived molecules with immunomodulatory properties are considered, and their therapeutic efficacy in models of autoimmunity and allergy described. The recent and future use of nematodes and their products in treating human disease are also discussed.

ES-62 is a secreted protein of filarial nematodes that possesses multiple immunomodulatory activities. A full characterization of these activities awaits elucidation but to date it has been shown that ES-62 can inhibit pro-inflammatory/Th1 immune responses and in some studies, it has been found to actively support Th2 development. As an active filarial nematode infection is associated with a Th2-like immunological phenotype, this study investigated whether ES-62 was likely to be responsible for, or at least contribute to, this phenotype. Specifically, we determined ES-62's effect on the immune response to two other filarial nematode antigens, chosen for their ability to promote Th1 responses. The two antigens were recombinant Onchocerca volvulus-Fatty acid And Retinol-binding-1 (rOv-FAR-1) and recombinant Onchocerca volvulus-Activation associated Secreted Protein-1 (Ov-ASP-1). Overall the results show that in spite of its previously characterized immunomodulatory properties, ES-62 was unable to modulate/reverse the Th1 immune responses induced by the two Onchocerca antigens. Therefore, in this study no support is provided for the idea that ES-62 might be a major player in facilitating the overall immunological phenotype in filariasis and reasons for this somewhat surprising outcome are discussed.

There is currently great interest in the idea of using helminth-derived molecules for therapeutic purposes and indeed we have shown that ES-62, a filarial nematode-derived phosphorylcholine-containing glycoprotein, significantly reduces the severity of arthritis in a murine model. Clearly, knowledge of mechanism of action is important when considering molecules for use in treating disease and although much is known regarding how ES-62 interacts with the immune system, gaps in our understanding remain. A feature of filarial nematode infection is a defective, T helper 2 (Th2)-polarized antigen-specific T-cell response and in relation to this we have recently shown that ES-62 inhibits clonal expansion and modulates effector function towards a Th2 phenotype, of antigen-specific T cells in vivo. ES-62 is also known to directly modulate B-cell behaviour and hence to determine whether it was mediating these effects on T cells by disrupting B-T-cell co-operation, we have investigated antigen-specific responses using an adoptive transfer system in which traceable numbers of tg ovalbumin (OVA)-specific T cells and hen egg lysozyme (HEL)-specific B cells respond to a chemically coupled form of OVA-HEL that contains linked epitopes that promote cognate T- and B-cell interactions. Surprisingly, these studies indicate that activated B cells restore T-cell expansion and prevent Th2-like polarization. However, ES-62-treated double cell transfer mice demonstrate a more generalized immunosuppression with reduced levels of Th1 and -2 type cytokines and antibody subclasses. Collectively, these results suggest that whilst ES-62 can target B-T-cell co-operation, this does not promote polarizing of T-cell responses towards a Th2-type phenotype.

Characterization of the immune response to Onchocerca volvulus is important for the diagnosis, control and understanding of the disease it causes. The antibody response to surface, secreted and somatic antigens of the worm has therefore been examined at an individual immunoglobulin (Ig) class level, by using a panel of different human sera. Onchocerca-specific antigens tend to be of low molecular mass and preferentially recognized by IgG4 and IgE. There is considerable cross-reaction between O. volvulus and O. gibsoni, so that the latter may be an alternative source of material for use in diagnosis. A surface-enriched fraction of low molecular mass appears to be a most promising diagnostic tool. Amongst somatic antigens, two were uniquely recognized by IgG3 antibodies in sera from sowda patients, thereby providing a molecular correlate for a recognized pathological condition. Improved diagnosis is needed for detecting infection in both humans and the vector. Our target for detection in humans is a continuously released, nonimmunogenic product, which is ideally stage and parasite specific. The excretions of adult worms do contain components not recognized by antibodies in infected serum, but we cannot rule out that these are of host, rather than parasite origin. Excretions of Litomosoides carinii contain both host and parasite molecules and, in addition, stage-specific and sex-specific components. Unfortunately, however, the rate of production of excretions varies during the life of L. carinii. This finding may be relevant to the detection of Onchocerca excretions if they are produced at a similarly uneven rate. Finally, for detecting infective larvae in the vectors, we are currently screening a genomic library of O. volvulus for an appropriate probe. To date, one DNA sequence has been cloned that shows promising specificity.

Objective:In countries where parasitic infections are endemic, autoimmune disease is relatively rare, leading to the hypothesis that parasite-derived immunomodulators may protect against its development. Consistent with this, we have previously demonstrated that ES-62, a 62 kDa phosphorylcholine (PC)-containing glycoprotein that is secreted by filarial nematodes, can exert anti-inflammatory action in the murine collagen-induced arthritis (CIA) model and human rheumatoid arthritis-derived synovial tissue cultures. As a first step to developing ES-62-based drugs, the aim of this study was to determine whether the PC-moiety of ES-62 was responsible for its anti-inflammatory actions.Methods:We compared the anti-inflammatory activity of a PC-free form of recombinant ES-62 (rES-62) and a synthetic PC-ovalbumin conjugate (OVA-PC) with that of native ES-62 in the CIA model and synovial tissues from patients with rheumatoid arthritis.Results:The anti-inflammatory actions of ES-62 in CIA appear to be dependent on the PC moiety as indicated by the reduction in severity of disease and also suppression of collagen-specific T helper 1 cytokine production observed when testing OVA-PC, but not rES-62. Interestingly, the anti-inflammatory activity of PC did not correlate with a reduction in anti-collagen IgG2a levels. Also, the ES-62-mediated suppression of interferon-γ from human patient tissues could be mimicked by OVA-PC but not rES-62 or ovalbumin.Conclusions:In countries where filariasis is endemic the reduced detection of inflammatory diseases, such as rheumatoid arthritis may be because of the anti-inflammatory action of the PC moieties of ES-62. PC may thus provide the starting point for the development of novel, safe immunomodulatory therapies.

An unusual feature of nematodes is the covalent attachment of immunomodulatory phosphorylcholine (PC) moieties to N-type glycans. Our previous work on the filarial nematode glycoprotein ES-62 has enabled us to predict the identity of enzymes necessary for PC-N-glycan biosynthesis. Here, we addressed these predictions using gene knockout technology applied to C. elegans and present two pieces of confirmatory data. Employing a triple null mutant worm lacking all three genes that encode active UDP-N-acetyl-D-glucosamine: alpha-3-D-mannoside beta1, 2-N-acetylglucosaminyltransferase I (GnT I) we have confirmed our earlier prediction that a crucial step in the generation of the substrate for PC transfer is addition of terminal GlcNAc to the alpha1-3-linked mannose residue of the glycan by GnT I. Second, by silencing genes responsible for expressing enzymes of the Kennedy pathway of phosphatidylcholine biosynthesis by RNA interference (RNAi), we have confirmed our belief for a role for diacylglycerol: choline phosphotransferase (CPT) in PC-N-glycan biosynthesis.

SUMMARY Epidemiological evidence has led to the hypothesis that the concurrent and predominant transmission of Onchocerca ochengi by Simulium damnosum s.l. in sub-Saharan Africa could lead to the protection of humans against onchocerciasis caused by Onchocerca volvulus (zooprophylaxis). To gain support for this hypothesis, we investigated whether exposure to O. volvulus could protect cattle from O. ochengi. Gudali calves were vaccinated with live O. volvulus -infective larvae and subsequently challenged with O. ochengi-infective larvae whilst raised in a fly-proof house. Post-challenge adult parasite and microfilaria development, IgG1 and lgG2 subclass antibodies response to Ov10/Ov11 recombinant Onchocerca antigens, and peripheral blood lymphocyte proliferative responses to O. ochengi crude antigens were studied over a 1-year period. The vaccinated-challenged animals had 83 – 87% less adult O. ochengi parasites than non-vaccinatedchallenged animals. IgG1 and lgG2 antibodies to Ov10/Ov11 recombinant Onchocerca antigens were invoked by nonvaccinated-challenged animals but not by most (80%) of the vaccinated-challenged animals. These findings support the idea of cross-protection (zooprophylaxis) due to inoculation of humans with O. ochengi-infective larvae under natural transmission conditions in endemic areas.

The plant 2S seed albumins Ber e 1 and SFA8, although structurally very similar, vary with respect to their allergenic properties. Whereas the former represents a major allergen, the latter appears to promote only weak allergenic responses. The aim of this investigation was to determine whether the allergenic properties of Ber e 1 and SFA8 reflected differential polarization of dendritic cell (DC) and Th cell responses. We thus investigated the effect of recombinant forms of both allergens on DC and Th cell responses as indicated by cell surface phenotype and cytokine production. Exposure of murine DCs to SFA8, but not Ber e 1, resulted in production of the cytokines IL-12 p40 and TNF-α by a mechanism independent of recognition by TLRs. Furthermore, depending on the mouse strain used, increased expression of MHC class II and costimulatory molecules such as CD40, CD80, and CD86 was associated with exposure to SFA8, but not Ber e 1. In coculture experiments using the DO11.10 transgenic T cell that recognizes OVA peptide, DCs exposed to both allergens induced T cells to produce IFN-γ, but only Ber e 1 could induce significant production of IL-4 and IL-5. Likewise, analysis of transcription factors shows increased T-bet with respect to both allergens, but also GATA-3 with respect to Ber e 1. Overall, our data are consistent with the idea that the ability of Ber e 1, but not SFA8, to act as a potent allergen may reflect differences in their ability to induce IL-12 production.

SUMMARY We have previously shown glycosphingolipids of Ascaris suum to have phosphorylcholine (PC) and non-PC immunomodulatory moieties. In the present study we further investigated the nature of the immunomodulatory moieties by employing three synthetic glycosphingolipids each possessing features of the original molecule to examine effects on macrophage and dendritic cell (DC) cytokine production and surface co-stimulatory molecule expression. Compound 2, which lacked PC but contained ceramide, had no effect on either macrophages or DCs. Surprisingly however, Compound 1, which contained PC and hence arguably most resembled the native material, had, with the exception of a small increase in surface antigen expression, no immunomodulatory properties. Conversely, Compound 3, which contained PC but was otherwise least like the native molecule, demonstrated a number of effects on both macrophages and DCs, including induction of Th-1/pro-inflammatory cytokines, inhibition of such cytokines induced by IFN-γ/LPS and increased expression of co-stimulatory molecules. Taken together these results indicate: (i) that although PC is an immunomodulatory component of the native molecule other structural feature are necessary to allow it to act; (ii) that carbohydrate rather than ceramide is likely to represent a non-PC immunomodulatory moiety; and (iii) that synthetic PC-containing molecules have the potential to act as immunomodulatory drugs.

ES-62, a filarial nematode-derived anti-inflammatory immunomodulator, was administered to mice in an attempt to prevent pathology associated with Toxoplasma gondii infection. The nematode product was shown to elevate mitogen and T. gondii-specific IL-10 production but was unable to inhibit Th1 responses. Consequently ES-62 could not prevent Th1 generated immunopathology.

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-α, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-γ. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-α production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.

We have previously shown in an in vitro study that the filarial nematode phosphorylcholine (PC)-containing glycoprotein ES-62 promotes a murine dendritic cell (DC) phenotype that induces T helper type 2 (Th2) responses. We now show that, in addition to directly priming Th2 responses, ES-62 can act to dampen down the pro-inflammatory DC responses elicited by lipopolysaccharide. Furthermore, we also demonstrate that murine DCs and macrophages derived ex vivo from bone marrow cells exposed in vivo to ES-62 by release from osmotic pumps are hyporesponsive to subsequent stimulation with lipopolysaccharide. These effects can be largely mimicked by exposure to the PC moiety of ES-62 conjugated to an irrelevant protein. The data we provide are, as far as we aware, the first to show that a defined pathogen product can modulate the developmental pathway of bone marrow cells of the immune system in vivo. Such a finding could have important implications for the use of pathogen products or their derivatives for immunotherapy.

Mice were subcutaneously implanted with osmotic pumps loaded with ES-62, an immunomodulatory phosphorylcholine (PC)-containing glycoprotein secreted by filarial nematodes. The concentration of ES-62 was set to give a serum level within the range found for PC-containing molecules during natural filarial nematode infection of humans. Peritoneal B1 cells were recovered from the mice and the effect of exposure to ES-62 on a number of parameters determined ex vivo. B1 cells exposed to ES-62 showed an increase in spontaneous proliferation that was enhanced by ex vivo exposure to F(ab')(2) fragments of anti-IgM antibodies (anti-IgM), to activate via the antigen receptor, or LPS. Consistent with this, cell-cycle analysis indicated that cells pre-exposed to ES-62 showed increased cell-cycle progression following stimulation with anti-IgM. Pre-exposed cells also showed an increase in both spontaneous and anti-IgM induced IL-10 secretion. Taken together, these data indicate that ES-62 activates murine B1 cells in vivo. Conversely, we have previously shown conventional (B2) B cells to be rendered hypo-responsive by in vivo exposure to ES-62 and the different effect on the two cell types is discussed in relation to the nature of the antibody response arising during filarial nematode infection.

Understanding modulation of the host immune system by pathogens offers rich therapeutic potential. Parasitic filarial nematodes are often tolerated in human hosts for decades with little evidence of pathology and this appears to reflect parasite-induced suppression of host proinflammatory immune responses. Consistent with this, we have previously described a filarial nematode-derived, secreted phosphorylcholine-containing glycoprotein, ES-62, with immunomodulatory activities that are broadly anti-inflammatory in nature. We sought to evaluate the therapeutic potential of ES-62 in vitro and in vivo in an autoimmune disease model, namely, collagen-induced arthritis in DBA/1 mice. ES-62 given during collagen priming significantly reduced initiation of inflammatory arthritis. Crucially, ES-62 was also found to suppress collagen-induced arthritis severity and progression when administration was delayed until after clinically evident disease onset. Ex vivo analyses revealed that in both cases, the effects were associated with inhibition of collagen-specific pro-inflammatory/Th1 cytokine (TNF-α, IL-6, and IFN-γ) release. In parallel in vitro human tissue studies, ES-62 was found to significantly suppress macrophage activation via cognate interaction with activated T cells. Finally, ES-62 suppressed LPS-induced rheumatoid arthritis synovial TNF-α and IL-6 production. Evolutionary pressure has promoted the generation by pathogens of diverse mechanisms enabling host immune system evasion and induction of “tolerance.” ES-62 represents one such mechanism. We now provide proof of concept that parasite-derived immunomodulatory strategies offer a novel therapeutic opportunity in inflammatory arthritis.

Bioactive interleukin (IL)-12 is a 70 000-molecular weight (MW) heterodimeric cytokine comprising p40 and p35 chains. However, p40 can also form homodimers that antagonize bioactive IL-12 or heterodimerize with p19 to form IL-23, which exhibits overlapping yet distinct functions to that of IL-12. We now define distinct signalling mechanisms that regulate lipopolysaccharide (LPS)-mediated induction of IL-12 p40 and p35 in macrophages and which may therefore provide therapeutic targets for precise and specific fine-tuning of cytokine responses. Thus, whilst LPS-induced p38 mitogen-activated protein kinase (MAPkinase) activation is required for the induction of both p40 and p35 subunits, Erk MAPkinase signalling mediates negative feedback regulation of p40, but not p35, production. Such Erk MAPkinase activation is downstream of calcium influx and targets LPS-induced IL-12 p40 transcription by suppressing the synthesis of the transcription factor, interferon regulatory factor-1 (IRF-1). In contrast, negative regulation of the p35 subunit of IL-12 occurs via a calcium-dependent, but Erk-independent, mechanism, which is likely to involve nuclear factor (NF)-kappa B signalling. Finally, the importance of both Erk and p38 MAPkinases in differentially regulating IL-12 p40 and p35 production is underscored by each being targeted by ES-62, a product secreted by parasitic filarial nematodes to polarize the immune system towards an anti-inflammatory phenotype conducive to their survival.

MyD-1 (CD172) is a member of the family of signal regulatory phosphatase (SIRP) binding proteins, which is expressed on human CD14+ monocytes and dendritic cells. We now show a novel role for MyD-1 in the regulation of the innate immune system by pathogen products such as lipopolysaccharide (LPS), purified protein derivative (PPD), and Zymosan. Specifically, we demonstrate that ligation of MyD-1 on peripheral blood mononuclear cells (PBMCs) inhibits tumor necrosis factor alpha (TNFα) secretion but has no effect on other cytokines induced in response to each of these products. In an attempt to understand the molecular mechanisms underlying this surprisingly selective effect we investigated signal transduction pathways coupled to MyD-1. Ligation of the SIRP was found to recruit the tyrosine phosphatase SHP-2 and promote sequential activation of phosphatidylinositol (PI) 3-kinase, phospholipase D, and sphingosine kinase. Inhibition of LPS-induced TNFα secretion by MyD-1 appears to be mediated by this pathway, as the PI 3-kinase inhibitor wortmannin restores normal LPS-driven TNFα secretion. MyD-1-coupling to this PI 3-kinase-dependent signaling pathway may therefore present a novel target for the development of therapeutic strategies for combating TNFα production and consequent inflammatory disease. (Blood. 2003;102:2532-2540)

ES-62, a protein secreted by filarial nematodes, parasites of vertebrates including humans, has an unusual posttranslational covalent addition of phosphorylcholine to an N-type glycan. Studies on ES-62 from the rodent parasite Acanthocheilonema viteae ascribe it a dominant role in ensuring parasite survival by modulating the host immune system. Understanding this immunomodulation at the molecular level awaits full elucidation but distinct components of ES-62 may participate: the protein contributes aminopeptidase-like activity whereas the phosphorylcholine is thought to act as a signal transducer. We have used biophysical and bioinformatics-based structure prediction methods to define a low-resolution model of ES-62. Sedimentation equilibrium showed that ES-62 is a tightly bound tetramer. The sedimentation coefficient is consistent with this oligomer and the overall molecular shape revealed by small angle x-ray scattering. A 19Å model for ES-62 was restored from the small-angle x-ray scattering data using the program DAMMIN which uses simulated annealing to find a configuration of densely packed scattering elements consistent with the experimental scattering curve. Analysis of the primary sequence with the position-specific iterated basic local alignment search tool, PSI-BLAST, identified six closely homologous proteins, five of which are peptidases, consistent with observed aminopeptidase activity in ES-62. Differences between the secondary structure content of ES-62 predicted using the consensus output from the secondary structure prediction server JPRED and measured using circular dichroism are discussed in relation to multimeric glycosylated proteins. This study represents the first attempt to understand the multifunctional properties of this important parasite-derived molecule by studying its structure.

Filarial nematodes produce proteins containing phosphorylcholine (PC) covalently attached to N-linked glycans. Our previous work has suggested that transfer of PC might be dependent on a metabolite of the Kennedy pathway of phospholipid biosynthesis. In this study we have investigated whether the end product of this pathway, phosphatidylcholine, and in addition, sphingomyelin, could act as PC donors. Pulse-chase experiments employing [3H]choline as radiolabel, ruled out sphingomyelin, as the Acanthocheilonema viteae PC-containing protein, ES-62, was radiolabelled 20–30 min prior to the lipid. Phosphatidylcholine however was labelled immediately before ES-62 increasing the possibility that it could act as donor. This was further investigated by radiolabelling phosphatidylcholine synthesised via an alternative pathway such that other metabolites in the Kennedy pathway were not labelled. Specifically, we labelled the choline component of phosphatidylcholine using both [3H]serine and [3H]S-adenosyl methionine (SAM). Incubation of worms with [3H]serine failed to result in labelling of the PC component of ES-62 whereas the presence of [3H]SAM in the medium led to labelling of ES-62 but only 24 h after labelling of phosphatidylcholine. As ES-62 is labelled within minutes of phosphatidylcholine when employing [3H]choline as radiolabel, this suggests that labelling of ES-62 when using SAM is not due to direct transfer of PC from phosphatidylcholine. It is therefore concluded that neither sphingomyelin nor phosphatidylcholine act as PC donors for filarial nematode glycoproteins. The analysis of PC-containing metabolites and products from A. viteae additionally revealed the presence of PC-substituted glycolipids that were also radiolabelled by the use of [14C]choline. The kinetics of radiolabelling however differed from that observed in the case of ES-62, sphingomyelin and phosphatidylcholine in so far as labelled glycolipids were first detectable hours rather than minutes after addition of [14C]choline.

Parasite survival and host health may depend on the ability of the parasite to modulate the host immune response by the release of immunomodulatory molecules. Excretory-secretory (ES)-62, one such well-defined molecule, is a major secreted protein of the rodent filarial nematode Acanthocheilonema viteae, and has homologues in human filarial nematodes. Previously we have shown that ES-62 is exclusively associated with a Th2 Ab response in mice. Here we provide a rationale for this polarized immune response by showing that the parasite molecule suppresses the IFN-γ/LPS-induced production, by macrophages, of bioactive IL-12 (p70), a key cytokine in the development of Th1 responses. This suppression of the induction of a component of the host immune response extends to the production of the proinflammatory cytokines IL-6 and TNF-α, but not NO. The molecular mechanism underlying these findings awaits elucidation but, intriguingly, the initial response of macrophages to ES-62 is to demonstrate a low and transient release of these cytokines before becoming refractory to further release induced by IFN-γ/LPS. The relevance of our observations is underscored by the finding that macrophages recovered from mice exposed to “physiological” levels of ES-62 by the novel approach of continuous release from implanted osmotic pumps in vivo were similarly refractory to release of IL-12, TNF-α, IL-6, but not NO, ex vivo. Therefore, our results suggest that exposure to ES-62 renders macrophages subsequently unable to produce Th1/proinflammatory cytokines. This likely contributes to the generation of immune responses with an anti-inflammatory Th2 phenotype, a well-documented feature of filarial nematode infection.

Unraveling the molecular mechanisms by which filarial nematodes, major human pathogens in the tropics, evade the host immune system remains an elusive goal. We have previously shown that excretory-secretory product-62 (ES-62), a homologue of phosphorylcholine-containing molecules that are secreted by human parasites and which is active in rodent models of filarial infection, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal transduction elements in B lymphocytes. Such activation mediates desensitization of subsequent B cell Ag receptor (BCR) ligation-induced activation of extracellular signal-regulated kinase-mitogen-activated protein (ErkMAP) kinase and ultimately B cell proliferation. We now show that the desensitization is due to ES-62 targeting two major regulatory sites of B cell activation. Firstly, pre-exposure to ES-62 primes subsequent BCR-mediated recruitment of SHP-1 tyrosine phosphatase to abolish recruitment of the RasErkMAP kinase cascade via the Igαβ-ShcGrb2Sos adaptor complex interactions. Secondly, any ongoing ErkMAP kinase signaling in ES-62-primed B cells is terminated by the MAP kinase phosphatase, Pac-1 that is activated consequently to challenge via the BCR.

International audience; The intensity of natural transmission of Onchocerca ochengi and Onchocerca volvulus by anthropo-boophilic Simulium damnosum s.l. was studied longitudinally in two cattle watering sites of a cattle ranch within a predominantly cattle populated area of the Guinea savanna of Cameroon and related to cattle O. ochengi skin microfilaria abundance. During the 12 months study period, a total of 4 696 flies was individually dissected to examine the monthly transmission potential (MTP) of O. ochengi and O. volvulus. The estimated Simulium damnosum s.l. annual biting rates (ABR) on human baits were 47 529 flies at the bank of the Vina "du sud"river. The ABR at the lake, which was situated at about 2 km upland from the perennial river, was 8 579. The monthly parous rate was highly correlated with monthly biting rate. The annual transmission potentials (ATP) of O. ochengi were calculated to be 7 732 and 1 669 at the riverbank and the lake, respectively. Transmission occurred mainly in the dry season, peaking in the months of January to mid-March when dermal microfilaria density in the animals was also the highest. The O. ochengi microfilaria uptake by the fly vectors was host microfilaria density-dependent. The MTP of O. ochengi was positively correlated with dermal microfilaria density. The mean number of microfilariae per fly taken up during a blood meal was high during the dry season as was the mean number of infective larvae per fly but declined significantly with the onset of the early rains. A similar seasonality of transmission was also observed for O. volvulus that was concurrently transmitted by the same vector flies, but its ATP was comparatively much lower: 1 332 infective larvae per man per year at the riverbank and 107 around the lake. The population dynamics of cattle microfilariae therefore plays an important role in the regulation of O. ochengi transmission.; Dynamique de la transmission de Onchocerca ochengi et corrélation entre la densité de leurs microfilaires chez les bovins et le potentiel de transmission. L'intensité de la transmission naturelle de Onchocerca ochengi et Onchocerca volvulus par l'anthropo-boophile Simulium damnosum s.I. a été étudiée longitudinalement à deux points d'eau d'un élevage de bovins, situé dans une région de savane guinéenne du Cameroun comportant une population principalement bovine, et a été corrélée à l'abondance en microfilaires de O. ochengi dans la peau des bovins. Au cours des 12 mois de l'étude, un total de 4 696 mouches a été disséqué individuellement, afin d'examiner le potentiel de transmission mensuel (PTM) de O. ochengi et O. volvulus. Les taux annuels de morsures (TAM) estimés de Simulium damnosum s.I. sur des proies humaines étaient de 47 529 mouches sur les berges de la rivière Vina du Sud. Les TAM autour du lac, situé à environ 2 km sur les plateaux dominant la rivière permanente, était de 8 579. Le taux de parité mensuelle était fortement corrélé au taux mensuel de morsures. Les potentiels de transmission annuels (PTA) de O. ochengi ont été de 7 732 et 1 669 au niveau de la rivière et du lac, respectivement. La transmission s'est produite principalement pendant la saison sèche, avec un pic pendant les mois de janvier à mi-mars, lorsque la densité de microfilaires dans le derme des animaux était également la plus forte. Le captage des microfilaires de O. ochengi par les mouches vecteurs était dépendant de la densité en microfilaires dans le derme de l'hôte. Le PTM de O. ochengi était positivement corrélé à la densité en microfilaires du derme. Le nombre moyen de microfilaires capté par mouche durant un repas sanguin était élevé durant la saison sèche, comme l'était le nombre moyen de larves infestantes par mouche, mais déclinait significativement avec le début des premières pluies. Un phénomène saisonnier similaire a été également observé pour la transmission de O. volvulus, qui était simultanément transmis par les mêmes mouches vecteurs, mais son PTA était comparativement beaucoup plus faible : 1 332 larves infestantes par humain et par an au bord de la rivière, et 107 autour du lac. La dynamique de population des microfilaires des bovins joue donc un rôle important dans la régulation de la transmission de O. ochengi.

Publisher Summary This chapter focuses on immunomodulatory phosphorylcholine (PC)-containing proteins secreted by filarial nematodes. The filariids are arthropod-transmitted nematode worms that parasitize a wide range of vertebrates including man. Infection in humans is long-term and hundreds of millions of people are infected with these parasites. The longevity of these worms is readily explained by the immunomodulation theory as “defects” in immune responsiveness are revealed in infected individuals. Some discrepancy exists as to the exact nature of these defects but generally, they incorporate impairment of lymphocyte proliferation and bias in production of both cytokines. All filarial nematodes examined to date have been found to actively secrete proteins that contain a PC moiety. These molecules can be found in the bloodstream of individuals infected with filarial nematodes. The immunomodulatory properties of PC on filarial nematode molecules are elaborated in the chapter. PC capability of inhibiting lymphocyte proliferation by modulating associated effects on signal transduction pathways is described and mechanisms of action of PC are explained. Attachment of PC to filarial molecules as a target for chemotherapy is also discussed.

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein which is secreted by the rodent filarial nematode Acanthocheilonema viteae. A homologue exists in the human filarial nematode Brugia malayi and indeed PC is found attached to glycoproteins of many, if not all, filarial species. At concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans, ES-62 is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B and T lymphocytes following in-vitro exposure. Although this interaction is insufficient to cause lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways and hence also to proliferation via the antigen receptors. The active component of ES-62 appears to be PC, as the results obtained with ES-62 are broadly mimicked by PC conjugated to BSA or PC alone. Although PC can also be shown to desensitize B cells following in-vivo administration, not all cells are affected, as it is still possible to generate an antibody response. Dissection of this response indicates that it is of the Th2 type.

Filarial nematodes constitute major causes of morbidity in the Tropics. The worms have a life-span exceeding five years, a longevity which is considered to reflect at least in part, their ability to interfere with host lymphocyte responsiveness. To date the molecular mechanisms underlying this ability have not been defined but we now demonstrate that ES-62, a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to render Jurkat T cells anergic to intracellular signalling via the antigen receptor (TCR). In particular, ES-62 acts by modulating activation of the tyrosine kinases Fyn, Lck and ZAP-70 leading to selective disruption of TCR coupling to the phospholipase D, protein kinase C, phosphoinositide-3-kinase and RasMAPkinase signalling cascades. These cascades are key elements in the transduction of transcriptional and proliferative signals following ligation of TCR. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES may play a role in the induction of T lymphocyte hyporesponsiveness observed during filarial infections.

We have previously demonstrated by Western blot analysis that the adult stage of the filarial nematode Acanthocheilonema viteae expresses the alpha-subunits of heterotrimeric G-proteins corresponding to GS and Gq. We now show, using the same technique, that these two alpha-subunits are not detectable in the microfilaria stage of the parasite. Conversely, microfilariae contain Go, an alpha-subunit not expressed by the adult worm. No other G-protein alpha-subunits were found in microfilariae by Western blotting. However, reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate G-protein oligonucleotide primers, followed by hybridisation analysis, using oligonucleotides specific for individual G-protein alpha-subunits, not only confirmed expression of Go, but also detected Gi1 and G11 alpha-subunits. G-protein expression in infective larvae was also investigated by RT-PCR analysis: this stage of the organism was found to resemble the adult more than the microfilaria but differed from the adult in that GS was absent and Gi3 was present. The significance of these stage-specific differences in G-protein expression is discussed with respect to their possible role in parasite development and survival.

The molecular mechanisms underlying B- and T-cell development are, as yet, poorly understood. However, as G proteins regulate a diverse range of biological responses including growth, proliferation and differentiation, we have investigated differential expression of G proteins during B- and T-cell development with the aim of identifying key signals involved in lymphocyte maturation. Differential expression of beta 1/2 and alpha-subunits of the Gs-, i- and q-families was found throughout lymphoid development. Most strikingly, G alpha i1 and G alpha i1 were very weakly, or not expressed in pre-, immature and mature B cells, thymocytes or mature T cells, but strongly induced in mature B-lymphoblastoid cell lines, some of which have been used as models of germinal centre B cells, suggesting that expression of these G proteins may correlate with the later stages of B-cell development. In contrast, G alpha 16 expression was highest in T cells and pre-B cells and progressively declined with B-cell maturation. These findings suggest that G proteins, and the signals they regulate, such as ion channels and/or adenylate cyclase (G alpha s/i) and phospholipase C (G beta gamma and G alpha 11/16) are differentially regulated in lymphoid cells in a maturation-and lineage-dependent manner.

The mechanism of linkage of phosphorylcholine (PC) to excretory–secretory products (ES) of adult Brugia pahangi has been investigated. Biosynthetic radio-isotope labelling of ES with [3H]choline followed by SDS–PAGE/fluorography revealed a smear of molecular weight approximately 40–100 kDa which loses its radiolabel following exposure to N-glycosidase F, but not mild alkali. PC is thus attached to this smear of molecules via N-type glycans, a mechanism of linkage previously observed with respect to PC–ES of Acanthocheilonema viteae. Western blotting analysis of non-radiolabelled ES demonstrated the existence of additional PC–ES which were insensitive to N-glycosidase F, but not to alkali. This second group of molecules is therefore likely to contain PC linked to O-glycans. Filarial nematodes may thus utilize 2 classes of glycan for attachment of PC. Examination of B. pahangi and A. viteae whole worm extracts by Western blotting indicated that their PC content could not be cleaved by N-glycosidase F and hence the use of N-type glycans may be restricted to a subset of ES products. The implications of these findings with respect to developing inhibitors of PC attachment for use as anti-filarial drugs are discussed.

We have recently shown that the immunomodulatory substance phosphorylcholine (PC) is covalently attached to ES-62, a major secreted protein of the filarial nematode parasite Acanthocheilonema viteae, via an N-linked glycan. Linkage of PC to N-glycans is previously unreported, and hence we have investigated the biochemical events underlying it. PC addition was found by pulse-chase experiments to be a fairly early event during intracellular transport, occurring within 40-60 min of protein synthesis. Biosynthetic labeling/immunoprecipitation experiments revealed that addition of PC to ES-62 was blocked by (i) brefeldin A, an inhibitor of trafficking of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi, (ii) 1-deoxynorijirimycin, an inhibitor of glucosidase activity in the ER, and (iii) 1-deoxymannojirimycin, an inhibitor of mannosidase I in the cis Golgi. Swainsonine, an inhibitor of mannosidase II in the medial Golgi, did not affect PC addition. Taken together these data indicate that PC attachment is a post-ER event which is dependent on generation of an appropriate substrate during oligosaccharide processing. Furthermore, they strongly suggest that PC addition takes place in the medial Golgi and that the substrate for addition is the 3-linked branch of Man5GlcNAc3 or Man3GLcNAc3.

Adult Brugia pahangi were cultured with [3H]-choline in both the absence and the presence of either tunicamycin or 1-deoxymannojirimycin (dMM), inhibitors of N-linked glycosylation and N-linked oligosaccharide processing, respectively. Excretory-secretory products (ES) were recovered from the spent medium and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. Both inhibitors were found to prevent radiolabeling of ES, a result consistent with blockage of the addition of the highly immunodominant phosphorylcholine (PC) group. Tunicamycin/dMM-treated ES were subsequently employed to immunise a mouse in an attempt to produce monoclonal antibodies (mAbs) against non-PC epitopes of ES. Three mAbs were isolated, each of which reacted with ES but not with PC.

Parasitic worms skew immune responses to a Th2-like phenotype, inhibiting Th1/Th17 pathways and expanding regulatory cells such as Tregs. Worms can achieve such immunoregulation by secreting specific products like ES-62, a glycoprotein secreted by the filarial nematode A. viteae. We have previously shown ES-62 to be protective in Collagen-Induced Arthritis, a murine model of rheumatoid arthritis, and that the mechanism associated with protection involves a down-regulation of the cytokine IL-17. We have now analyzed the effects of ES-62 on other Th17-related cytokines focusing on IL-22, a cytokine that has been reported to exhibit both pro and anti-inflammatory actions in animal models of inflammatory disease. In CIA, administration of ES-62 appears to target the IL-17/IL-22 balance in lymph nodes and joints of animals resulting in modulation of the number and inflammatory phenotype of infiltrating and resident cells in the joint, and consequent attenuation of inflammation. Interestingly, using a combination of recombinant IL-22 and blocking antibody in in vivo studies, the precise type of effect of IL-22 on CIA progression was shown to depend largely both on the location of IL-22 action and also the stage at which the cytokine is produced during disease progression. These findings have allowed us to identify a novel mechanism used by the helminth product ES-62 to reduce autoimmune arthritis and they also contribute to our understanding of the complex biology of IL-22

The prevalence of autoimmunity in developing countries is lower than that in the developed world and this inverse relationship correlates with the level of chronic parasitic infections. Co-evolution of parasites with the human immune system has resulted in evasion strategies that prevent the host clearing the parasite yet limit pathology. One mechanism involves the release of immunomodulators like ES-62, secreted by the filarial nematode Acanthocheilonema viteae: ES-62 exhibits broad anti-inflammatory activity and therapeutic potential in autoimmune diseases such as rheumatoid arthritis. Here we show that ES-62 protects against nephritis, as indicated by proteinuria levels, in the murine MRL/Lpr model of systemic lupus erythematosus (SLE). It has been proposed that the pro-inflammatory cytokine IL-17 is a primary driver of disease, in mouse models as well as in SLE patients, but our data do not support targeting of IL-17 as the major protective mechanism underlying ES-62 efficacy. Indeed, and consistent with this, we have shown that neutralising antibodies specific for IL-17 do not block development of proteinuria. ES-62 is a large immunogenic glycoprotein and therefore not suitable for therapeutic use. Thus, since the active moiety of ES-62 is phosphorylcholine (PC), we have recently developed small molecular analogues based around PC that mimic the immunomodulatory effects of ES-62 in autoimmune disorders, including SLE.

Maintenance of adult male worms of the bovine filarial parasite Onchocerca gibsoni in vitro , in the presence of radioactive precursors, resulted in the time-dependent excretion-secretion of radiolabelled parasite macromolecules (ES). Molecules labelled with amino acids ([ 35 S] methionine, pH] leucine) covered a wide range of molecular weights, whereas labelling with [ 3 H] glucosamine produced predominantly molecules of high molecular weight. Many of the products were recognized by antibodies in two serum pools from humans infected with Onchocerca volvulus . O. gibsoni ES may therefore provide a substitute source of material for studies on the ES of the less readily available human parasite.

La structure de la PC-glycane du principal produit excretant-secretant (ES-62) de Acanthacheilonemo vitae a ete etudie grâce a l'utilisation d'endoglycasidases et de lectines. Les resultats obtenus developpent la possibilite qu'elle pourrait etre du type mannose. Cette hypothese et l'insensibilite de la PC-glycane aux traitements separant la PC au la choline des PC-glycanes bacteriennes, suggerent que cette structure serait plus proche des PC-glycanes fongiques que bacteriennes

E-S 62, a major excretory-secretory product of the rodent filarial parasite, Acanthocheilonema viteae, inhibits the polyclonal activation (DNA synthesis) of mouse B cells by mitogenic anti-Ig antibodies. This effect appears to be due at least in part to the phosphorylcholine (PC) moiety of the molecule, because it can be mimicked by PC-BSA or PC-chloride. Activation of the B cells by LPS is not influenced by the presence of E-S 62/PC, suggesting that they may target some aspect of signaling via the Ag receptor. E-S 62/PC failed to inhibit surface Ig-mediated generation of the second messenger, inositol triphosphate, indicating that their target may not be the early biochemical events associated with activation. Exposure to E-S 62/PC was found to lead to a reduction in the level of protein kinase C, an important downstream regulatory enzyme, in anti-Ig-treated cells. This protein-kinase C down-regulation may be the biochemical mechanism underlying E-S 62/PC-mediated inhibition of surface Ig-activated B cells.